Gamma-aminobutyric acid type A receptors (GABAARs) are the principal mediators of inhibitory transmission in the mammalian central nervous system. GABAARs can be localized at postsynaptic inhibitory specializations or at extrasynaptic sites. While synaptic GABAARs are activated transiently following the release of GABA from presynaptic vesicles, extrasynaptic GABAARs are activated continuously by resting concentrations and thus mediate tonic inhibition. These extrasynaptic GABAARs are predominantly composed of alpha-4 beta2/3 and gamma subunits, exhibit high affinity for GABA and have little or no desensitization. Steroid metabolites of progesterone and deoxycortisone (known as neurosteroids) have been shown to be potent positive allosteric modulators of extrasynaptic GABAA receptors. However the exact mechanisms by which neurosteroids alter extrasynaptic GABAARs function are not well understood. Previous experiments have suggested that Protein Kinase C (PKC) activity is required for neurosteroid-mediated modulation of GABAARs. We hypothesize that neurosteroids mediate PKC phosphorylation of extrasynaptic GABAARs to alter receptor function. In order to test this hypothesis, our laboratory has developed DNA constructs that were critical serines for PKC phosphorylation have been mutated to alanines (alpha4-S443A and beta-3-408/409A). I have first examined how these point mutations in PKC phosphorylation sites within alpha-4 and beta-4 subunits alter the neurosteroid-mediated changes in the magnitude of GABAergic currents. I have shown that the rundown of alpha4beta3-mediated GABAergic currents is prevented by phorbol ester stimulation of PKC and by the S443A mutation in the alpha-4 subunit. I will assess the physiological significance of mutations in PKC phosphorylation sites within the alpha-4 and beta-3 subunits and determine whether PKC-dependant phosphorylation plays a role in neurosteroid-mediated enhancement of tonic inhibition. These studies will utilize knock-in mice in which sites of neurosteroid-induced phosphorylation of GABAARs have been mutated, which will also allow us to examine the behavioral significance of this phenomenon. Neurosteroid levels are known to fluctuate during various physiological states and pathophysiological conditions. The work proposed in this grant will provide insight into neurosteroid-mediated alterations in neuronal excitability and might contribute to the development of novel pharmacological targets for the treatment of postpartum depression, epilepsy and anxiety disorders. PUBLIC HEALTH RELEVANCE: GABAA receptor subtypes that mediate tonic inhibition are uniquely sensitive to the actions of neurosteroids. Steroid-induced fluctuations in GABAA receptor subunit expression result in alterations in neuronal excitability and are implicated in syndromes such as postpartum depression and premenstrual syndrome. The studies in this application will provide insight into neurosteroid-mediated alterations in neuronal excitability an might contribute to the development of novel treatments for postpartum depression, epilepsy and anxiety disorders.